Search

We report a bottom-up synthetic biology approach to engineering vesicles with programmable permeabilities. Exploiting the concentration-dependent relationship between constitutively active pores (alpha-hemolysin) and blockers allows blockers to behave as molecular regulators for tuning permeability, enabling us to systematically modulate cargo release kinetics without changing the lipid fabric of the system.

This thesis is concerned with the development and application of AMPLE, a software pipeline for macromolecular crystallographic Molecular Replacement, to different classes of proteins. The ability of AMPLE to solve protein structures via Molecular Replacement was first explored with two new classes of proteins: coiled-coils and transmembrane helical proteins. The results were very positive, with AMPLE solving 75 of 94 (80%) of the coiled-coil and 10 of 15 (67%) of the transmembrane protein test cases. In both cases the performance of AMPLE was benchmarked against a library of ideal helices. The performance of idea helices was found to be surprisingly good (solving 44 of the coiled-coil and 7 of the transmembrane test cases), but the performance of AMPLE was significantly better. AMPLE's truncation and ensembling pipeline was then applied to the solution of protein structures using very distant homologs, and compared with the performance of the current state-of-the-art in automated Molecular Replacement in MRBUMP. The AMPLE pipeline was able to solve structures that could be be solved using MRBUMP, showing how AMPLE is able to find the evolutionarily conserved structural core from homologs that cannot be accessed using existing protocols. Work was also carried out to optimise AMPLE's cluster and truncate procedure. This has resulted in a significant improvement on AMPLE's ability to solve the structures in a difficult set of test cases (solving 11 of 18 test cases compared with 6 for the original protocol), despite only a modest increase in processing time. As part of this work, AMPLE has been extended from a prototype piece of software consisting of a collection of independent scripts, to a coherent, modularised program incorporating a range of software best practice. AMPLE is also

cADP-ribose (cADPR), a naturally occurring metabolite of NAD(+), has been shown to be an important regulator of intracellular Ca(2+) release. Considerable evidence suggests that cADPR is the endogenous modulator of the ryanodine receptor (RyR), which mediates Ca(2+)-induced Ca(2+) release (CICR). Indeed, cADPR-mediated Ca(2+) release is subject to functional regulation by other modulators of CICR, including Ca(2+), caffeine and calmodulin. However, the underlying basis behind the effect of such agents on cADPR activity (in particular whether they regulate cADPR binding), as well as the precise nature of the cADPR receptor remains unclear. In the present study, use of (32)P-radiolabelled cADPR has enabled a detailed pharmacological characterization of cADPR-binding sites in sea urchin egg homogenates. We report that cADPR binds specifically to a single class of high affinity receptor. Retainment of binding to membranes after a high-salt wash suggests the involvement of either an integral membrane protein (possibly the RyR itself) or a peripheral protein tightly associated to the membrane. Insensitivity of [(32)P]cADPR binding to either FK506 or rapamycin suggests that this does not concern the FK506-binding protein. Significantly, binding is highly robust, being relatively insensitive to both endogenous and pharmacological modulators of RyR-mediated CICR. In turn, this suggests that such agents modulate cADPR-mediated Ca(2+) release primarily by tuning the 'gain' of the CICR system, upon which cADPR acts, rather than influencing the interaction of cADPR with its target receptor. The exception to this is calmodulin, for which our results indicate an additional role in facilitating cADPR binding.

Sentinel node biopsy (SNB) has been promoted by claims of a survival advantage after early lymphadenectomy in patients with a positive result. The pivotal paper states that SNB “identifies patients with nodal metastases whose survival can be prolonged by immediate lymphadenectomy.”1 An earlier paper by Morton and colleagues states, “Our data suggest a significant therapeutic benefit for immediate …

It is a basic tenet of the sentinel lymph-node biopsy procedure that all positive sentinel lymph nodes will inevitably progress to palpable nodal recurrence if not removed. Comparison of survival is, therefore, considered permissible among patients with positive sentinel lymph nodes who undergo early lymphadenectomy with that among patients who have delayed lymphadenectomy for palpable regional node metastasis, providing that survival is calculated from the date of wide local excision of the primary tumor. Here, that fundamental assumption is contested and evidence is presented to show that a positive sentinel lymph node might have no adverse prognostic relevance in up to one-third of patients. Furthermore, in the same patients, progression to palpable nodal disease might not have occurred even if the positive sentinel node had not been removed. The term prognostic false-positivity is used to describe this phenomenon. Such patients are incorrectly up-staged, are given inaccurate prognostic information and can undergo unnecessary completion lymphadenectomy and unnecessary adjuvant therapy.

Sentinel node biopsy has been enthusiastically adopted into clinical practice without a clear understanding of its benefit, which is often assumed rather than proved. Recently, the status of the sentinel node has been shown to be the most important predictor of recurrence and survival for patients with malignant melanoma. The proportion of patients who have had a negative sentinel node biopsy and who are free of disease at three years is 88.5% compared with 55.8% of patients with a positive biopsy; these groups have overall survival rates of 93% and 67%, respectively.1 2 The thickness of the tumour and whether there is ulceration remain important prognostic discriminators in patients with a negative biopsy.1 But is the ability to stratify patients prognostically enough to justify the widespread use of sentinel node biopsy or should we wait for evidence of a survival benefit after selective node dissection in patients who have had a positive sentinel node on biopsy? Several reasons have been given to justify the use of sentinel node biopsy. Firstly, it is cheaper and causes less morbidity than elective lymph node dissection. The total cost per patient, including the cost of wide local excision, is $19 285 (£12 856) for elective lymph node dissection compared with $13 835 for sentinel node biopsy under …

We report a metastatic cutaneous melanoma of the medial rectus muscle of the right orbit which presented with diplopia, exophthalmos and retrobulbar pain. Radioimmunoscintigraphy using melanoma-specific monoclonal antibody was used to confirm the diagnosis and to show the presence of disseminated disease in other areas. Effective palliation was achieved with external beam irradiation.

The addition of tryptophan to a monoamine oxidase inhibitor (MAOI) has been recommended for patients failing to respond to an MAOI alone. The authors report acute behavioral and neurologic toxicity immediately after tryptophan administration in a patient taking phenelzine.

Summary: This study utilized isoelectric focusing and electrophoresis in an attempt to detect a cystic fibrosis (CF) scrum factor(s), for which there is considerable indirect evidence. The method of isoelectric focusing in polyacrylamide gels (IEFAG) specified by Wilson et al. (53), as reproduced in our laboratory, did not enable the detection of a CF factor protein reported to focus near pH 8.4–8.5. Consequently, we employed our modified IEFAG techniques, which enabled us to demonstrate significantly enhanced resolution and striking heterogeneity in serum γ-globulins. Despite the significant increase in the number of bands resolved by our methods, neither a difference at pH 8.4–8.5 nor other differences throughout the alkaline pH range could be detected consistently in the CF and heterozygous sera. The two-step IEFAG/disc electrophoresis technique outlined by Alt-land et al. (2), as reproduced in our laboratory, indicated that at least one small, cationic protein could be fractionated from all serum samples. Improvements in the method of disc electrophoresis resulted in the observation of numerous bands from some samples and of differences among the samples, but no protein band unique to the CF genotypes was observed. Our approach, employing different electrophoretic techniques and varying the conditions of sample analysis, should have increased the likelihood of detecting a protein or proteins specific for the CF genotypes. Despite the many variations in our approach, no consistently unique protein was observed in the CF or heterozygous sera. Speculation: A CF serum factor cannot be readily demonstrated by the electrophoretic techniques described. The value of the “biophysical assay” and the “nonbiologic technique” reported in the literature is suspect, and the promise and applicability of these techniques as diagnostic tests for the CF gene should be carefully evaluated.

The nauplii of the brine shrimp Artemia salina are dependent upon the function of their salt gland to maintain osmotic pressure within narrow limits. A number of drugs interfere with this function and are lethal to the nauplii. Saliva and serum from normal persons, patients with cystic fibrosis, and obligate heterozygotes were tested for lethal effect against brine shrimp nauplii. At salt concentrations between 100 mM and 2.5 no difference was found among the phenotypes. At lower concentrations a difference was noted occasionally between some normal subjects and some individuals carrying one or two genes for cystic fibrosis. Data from an independent series of experiments indicate that the naupliar deaths result from distorted ratios of Na+/K+ and not from a specific gene product. No difference was noted in the O2 uptake of nauplii treated with saliva or serum obtained from normal subjects, patients with cystic fibrosis, or obligate heterozygotes.